The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein.
Fatty acids, among which are included polyunsaturated fatty acids (abbreviated to PUFA), are particularly important biological compounds because they are involved in numerous biological processes such as building and maintenance of cell membranes, synthesis of hormones (for example, prostaglandins) that play a role in platelet aggregation, inflammation processes and in the human immunological response, etc.
The human body is able to synthesise most PUFAs, with the exception of two families of PUFAs known as essential fatty acids, which must necessarily be obtained through dietary intake.
The two families of essential fatty acids are:                omega-6, which are particularly abundant in nut oils, and oils derived from sunflower, soybean, grapeseed or corn, and in fatty poultry (such as duck);        omega-3 which are mostly found in nut oils, in plants such as rapeseed and flax, and in fatty fish (such as salmon, tuna, sardine, mackerel or herring). Omega-3 production processes using microalgae cultures, transgenic yeast or krill have recently been developed.        
Omega-3s are PUFAs that are considered particularly interesting for their antioxidant properties. Among these omega-3s, purified EPA (eicosapentaenoic acid, C20-5ω3) and DHA (docosahexaenoic acid, C22-6ω3) and enriched combinations thereof are the most frequently used as dietary supplements or medicinal products to help lower blood triglyceride levels, reduce cardiovascular risks, and improve vision or cognitive functions, etc.
Recent clinical studies have shown that treatment of patients having triglyceride levels above 500 ml/dL with 4 grams per day of 96% EPA ethyl ester without DHA made it possible to lower triglyceride levels, without increasing the levels of low density lipoproteins or LDL (“bad” cholesterol), whereas treatment with 4 grams per day of a mixture of ethyl esters of EPA and DHA, in proportions of about 50% and 35%, respectively, led to an increase in LDL levels along with the concomitant decrease in triglyceride levels.
Up to the present time, the PUFA dietary supplements used, in particular omega-3s, have essentially been based on mixtures containing 30% to 60% of a mixture of EPA and DHA. In the separation methods used to date, the mixture is obtained by means of transesterification of triglycerides into ethyl esters and subsequently through the enrichment of omega-3s by means of molecular distillation and/or co-crystallisation of the saturated and mono-unsaturated fatty acids with urea. Eventually the enriched ethyl esters are possibly reconverted into triglycerides by means of a chemical or preferably enzymatic process.
However, these separation methods are not satisfactory for the production of an omega-3 such as EPA, DHA or even stearidonic acid (SDA, C18-Sω3) at more than 80%, or even more than 96%, especially in esterified form.
In fact, the purification of omega-3s is complex because these compounds comprise a plurality of carbon-carbon double bonds which make them sensitive to oxidation or degradation. In the presence of oxygen and when they are heated, these PUFAs undergo reactions including in particular isomerisation, oxidation, peroxidation and oligomerisation.
Thus, the separation techniques indicated here above are used to obtain a mixture of PUFA with a good yield and an acceptable degree of purity; but they cannot be implemented for the individual separation of PUFAs. They thus do not allow for the separation of omega-3s from each other. Indeed, molecular distillation, for example, cannot economically eliminate DHA from EPA or SDA; it does not provide for an efficient separation of long chain omega-3s particularly of the C20 and C22 types. The combination of clathration with urea and molecular distillation provides the ability to obtain omega-3 mixtures of higher purity, at the expense of yield which is generally lower, and at high operating cost, but may not be used for the separation of long chain omega-3s from one another, and of EPA and DHA in particular.
There is therefore a need to provide a process for the industrial purification of omega-3 in ultra high purity esterified form.
Chromatography is a fine separation technique that allows for the efficient purification or enrichment of molecules under mild conditions and protected from light and air.
This technology is based on the separation of molecules that are brought into contact with a stationary phase with which they have different interactions. The use of one or more fluids, referred to as mobile phases or eluents, enables the percolation of various different molecules at different speeds. These different speeds allow for the physical separation of molecules and enable them to be harvested in purified form upon completion of the chromatographic processes using one or more columns. The purified fractions are in general concentrated under mild conditions at ambient or moderate temperatures, by means such as vacuum evaporation or membrane processes.
In some cases, the feedstock for the chromatographic purification is an oil composed of esters of fatty acids already enriched by molecular distillation, preferably comprising more than 30% of the omega-3 of interest, which has undergone a treatment process for the removal of oxidised compounds, either by means of the last molecular distillation, or by adsorption, preferably on silica derivatives (silica gel, bentonite, diatomaceous earth) or on activated carbon, for example.
Numerous prior art documents describe the elimination of oxidised compounds from compositions derived from oils.
By way of example, EP 0682006 discloses a process for treating oils containing omega-3s by dilution in hexane and addition of 10% to 40% by weight of active charcoal.
U.S. Pat. No. 4,874,629 discloses a process for treating oils containing omega-3s by steam distillation followed by adsorption of polar compounds on silica.
WO 2005/049 772 also describes a process for treatment of an oil rich in omega-3 by dissolution in an aprotic solvent and contacting with a silicon derivative.
EP 0773283 describes a process for treatment of an oil containing a PUFA with at least 18 carbons by contacting for at least 10 minutes with a minimum of 0.1% by weight of a diatomaceous earth pre-treated in an acid medium at a temperature of optionally 5° C. to 80° C., followed or not by steam distillation.
A fair number of chromatographic methods have also been described, for obtaining an omega-3 with high purity.
Thus, U.S. Pat. No. 5,719,302 discloses a method in which the PUFAs are separated in particular by means of a supercritical eluant (carbon dioxide under pressure), and in particular on a “Simulated Moving Bed”—SMB.
US 2011/0091947 describes another process for purification of omega-3 using the technique of simulated moving bed chromatography. The document describes in particular the succession of one step of enzymatic transesterification, two steps of molecular distillation, and one step of the SMB type, these last three steps making it possible to separate the products into two fractions in order of retention time.
WO 2011/080503 describes the purification of omega 3 using a system comprising two SMB chromatography devices arranged in series and a washing zone, each SMB chromatography device defining one separation zone and consisting of multiple columns. The feedstock to be treated is injected into a first separation zone to obtain an extract stream and a raffinate stream, said raffinate stream containing the compounds of interest being then injected into a column of the second separation zone that is not adjacent to a column of the first zone.
WO 2013/005051 describes the purification of omega-3 by means of two chromatographic separation processes by SMB or AMB (that is to say, “Actual Moving Bed”) in reversed phase mode with a water-organic eluent, wherein the two separations by SMB or AMB are performed sequentially on the same chromatographic device, or on two different devices, the intermediate purified by the first device being introduced into the second.
WO 2013/005048 describes the purification of EPA of over 90% purity by a first chromatographic separation followed by two chromatographic separations by SMB or AMB in reversed phase mode with a water-organic eluent in each step, the intermediate purified through the first chromatographic separation being introduced into the second chromatographic separation, and the intermediate purified through the second chromatographic separation being introduced into the third chromatographic separation.
In the two latter documents, the purified EPA is preferably obtained in the extract from the last separation; and thus the last separation separates EPA from the less retained impurities collected in the raffinate (as is well known to the person skilled in the art in reversed phase chromatography). Such impurities that are less retained than EPA may be, for example shorter chain PUFAs such as stearidonic acid (SDA), or oxidation compounds such as peroxides and aldehydes.
There is still a need to provide a PUFA (or a derivative thereof, in particular an ester thereof) with a high purity, allowing for its use in the preparation of medicinal compositions, from a multi compound feedstock including said PUFA. In particular, there is still a need to provide a PUFA (or a derivative thereof, in particular an ester thereof) that is essentially free of oxygenated contaminants such as peroxides or aldehydes.